Project Summary/Abstract Our mission in this program is to bring highly quantitative and high-content experimental and computational approaches to study the effects of space flight on the musculoskeletal system, focusing on cartilage, bone and synovium. The musculoskeletal disease focus is post-traumatic osteoarthritis, an all too common condition initiated in otherwise healthy (young to middle-aged) individuals who suffer a joint injury. The interactions between cartilage, bone and synovium in human joints are critically important for joint function and human motion on earth and in long-term space flight. Upon traumatic joint injury, there is an immediate upregulation of inflammatory cytokines in the synovial fluid that are secreted primarily by cells in the synovial membrane. When combined with mechanical trauma to cartilage accompanying joint injury, degradation of cartilage as well as subchondral bone often progresses to post-traumatic osteoarthritis. To study these interactions and how they may be ameliorated both on earth and in space, we propose to co-culture primary human explants of intact (native) cartilage, bone and synovial joint capsule tissue (obtained from a long-standing collaborating human donor bank). We will then test the effects of selected pharmacological agents (e.g., the anti-catabolic glucocorticoid, dexamethasone, and an anti-bone resorptive anti-sclerostin antibody, to prevent bone loss) to ameliorate tissue degradative processes. To perform these studies, our Aims are to validate an MPS model using osteochondral plugs co-cultured with joint capsule synovium, to challenge this model with inflammatory cytokines and an initial impact injury (associated with the early phases of post-traumatic OA), treat these challenged co-cultures with selected pharmacological agents on earth and in the international space station, and to address issues of patient stratification for treatment strategies, human donor variability, and response to therapeutics via biomarker discovery. Endpoint analyses include intracellular and extracellular biomarkers assessed using quantitative metabolomics and multiplexed protein release analyses. In addition, we will test the possibility that an optimized mechanical loading protocol on earth, post-flight, could have specific pro- anabolic and anti-catabolic effects on osteochondral tissues and a suppression of inflammatory cytokines from the synovium.